Elsevier

Molecular Immunology

Volume 127, November 2020, Pages 38-45
Molecular Immunology

Urokinase-type plasminogen activator receptor is required for impairing toll-like receptor 7 signaling on macrophage efferocytosis in lupus

https://doi.org/10.1016/j.molimm.2020.08.018Get rights and content

Highlights

Abstract

The accumulation of apoptotic cells is one of the pathological characteristics of systemic lupus erythematosus (SLE). The expression of urokinase-type plasminogen activator receptor (uPAR) has been reported to be increased in SLE patients and to be involved in macrophage efferocytosis. Although the toll-like receptor 7 (TLR7) is also over-expressed in lupus, its relationship to uPAR and its role in macrophage efferocytosis in lupus is still unclear. In the present study, we revealed that apoptotic cells accumulate in the spleen, macrophage efferocytosis is impaired, and uPAR is increased in the spleen and peritoneal macrophages of the TLR7 agonist imiquimod (IMQ)-induced SLE mouse model. Moreover, TLR7 upregulated uPAR expression in the mouse macrophage RAW 264.7 cells in vitro. The same results were also obtained using peritoneal macrophages of female Balb/c mice. When uPAR levels in peritoneal macrophages were knocked down by siRNA or inhibited by the peptide inhibitor UPARANT, and cells further treated with the TLR7 agonist R848, efferocytosis of peritoneal macrophages on apoptotic cells was restored. These results indicated that TLR7 activation impaired efferocytosis via uPAR in mouse peritoneal macrophages. Furthermore, TLR7 regulated uPAR expression via ERK/JNK signaling in macrophages. These results suggest that uPAR may be an important factor related to the accumulation of apoptotic cells in SLE.

Introduction

Efferocytosis refers to the clearance of apoptotic cells by phagocytes and plays an essential role in sustaining tissue homeostasis and regulating immune responses. Dying cells are removed either by tissue-resident professional phagocytes (such as macrophages and DCs) or by neighboring nonprofessional phagocytes (such as fibroblasts and endothelial cells) (Yurdagul et al., 2018). One of the common features of apoptotic cells is the increased surface exposure of the inner-membrane lipid phosphatidylserine (PtdSer) as a key 'eat-me' signal to attract phagocytes. At the plasma membrane of phagocytes, a series of bridging molecules including milk fat globule EGF factor 8 (MFGE8), protein S, and others are expressed. They can bind to PtdSer and are in turn recognized by surface receptors on phagocytes, such as αVβ3 integrin and Tryo3–Axl–Mer (TAM) family of receptors, to mediate efferocytosis (Poon et al., 2014). Under physiological conditions in adult humans, the rate of apoptotic cell removal is relatively high to ensure the continued clearance of approximately one million apoptotic cells per second (Park and Kim, 2017). Many diseases are associated with the non-clearance of apoptotic cells in tissues (Morioka et al., 2019). When apoptotic cells fail to be recognized and degraded, they may release autoantigens and activate autoreactive B cells to produce autoantibodies (Kimani et al., 2014).

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease that mainly affects women. The disease is characterized by the impairment of tolerance to self-antigens, increased production of auto-antibodies, and immune complex deposition in tissues, and it also implicates multiple organs (Urbonaviciute et al., 2019). Macrophages play an important role in the disease process (Yu et al., 2014). Lupus mice are characterized by increased toll-like receptor 7 (TLR7) expression in macrophages (Li et al., 2016), which may cause both imbalanced polarization and abnormal activation of macrophages. SLE patients show abnormalities in cell death at several levels, including increased rates of apoptosis, necrosis, and autophagy as well as reduced clearance of dying cells (Colonna et al., 2014). Impaired macrophage efferocytosis has been found in patients with SLE (Tsokos et al., 2016; Jung and Suh, 2015), which causes accumulation of cell debris and systemic inflammation (Ma et al., 2019). However, the specific mechanisms are still unclear.

The urokinase-type plasminogen activator receptor (uPAR/CD87) belongs to the lymphocyte antigen 6 protein superfamily and is expressed on many cell types including macrophages, monocytes, activated T cells, neutrophils, tumor cells, and endothelial cells (Enocsson et al., 2015). uPAR is anchored to the plasma cell membrane via a glycosyl phosphatidyl linositolanchor (GPI), and can be released by GPI-specific phospholipase C or D to a soluble form, namely suPAR (Smith and Marshall, 2010). Therefore, uPAR has no intracellular domain. It depends on interactions with other molecules, such as formyl peptide receptors (FPRs) (Cammalleri et al., 2016), vitronectin (Bae et al., 2013), and integrins (Alfano et al., 2009), to mediate intracellular signaling and be involved in cell adhesion and migration. At steady state, uPAR levels on the cell membrane and in serum are relatively stable (Laurenzana et al., 2019). However, inflammatory cytokines can upregulate them (Ni et al., 2016; Vasarhelyi, 2014). It has been reported that serum levels of suPAR, considered as a useful biological marker of SLE (Wen et al., 2018), were significantly increased in active lupus nephritis, and were associated with some clinicopathological features (Qin et al., 2015). Several studies have suggested that activation of TLRs (such as TLR2 (Liu et al., 2011) and TLR4 (Killeen et al., 2009)) leads to upregulation of monocyte/macrophage uPAR expression. However, there is no research on whether TLR7 (activating in SLE) regulates uPAR expression.

Given that uPAR can bind to several molecules associated with cell adhesion and phagocytosis, such as vitronectin and integrins, many studies have shown that uPAR is involved in efferocytosis. Park et al. (2009) have demonstrated increased macrophage efferocytosis of apoptotic neutrophils when uPAR was knocked out, and a wide range of integrins were involved in this event. Additionally, adding exogenous suPAR to the experimental system can inhibit efferocytosis. Conversely, Yang et al. (2014) have reported that there can be accumulation of cell debris in the spleen of uPAR-deficient mice after being injected with apoptotic cells. This suggests that uPAR is an essential factor in efferocytosis and facilitates it. Therefore, uPAR is linked to macrophage efferocytosis, but its role is still controversial. Based on the increased levels of serum suPAR (the soluble form of uPAR) in SLE and the impaired efferocytosis in SLE, we hypothesized that uPAR may be upregulated by TLR7 on macrophages and the elevated uPAR may inhibit efferocytosis.

Section snippets

Impaired macrophage efferocytosis and increased uPAR are shown in IMQ-SLE mice

To explore the relationship between macrophage efferocytosis and uPAR in SLE, we developed an SLE mouse model using the TLR7 agonist imiquimod (IMQ). After 4 weeks of treatment with imiquimod, mice exhibited a series of SLE symptoms. The levels of serum creatinine, anti-ds DNA, and IgG were higher in IMQ-SLE mice than in control mice (Fig. 1A, B, and C), and the weight of the spleen of IMQ-SLE mice was higher than that of the control mice (Fig. 1D). Histopathologic assessment of the kidneys

Discussion

Efferocytosis is critically important to maintain the homeostasis of organisms. Despite the clearance and degradation of apoptotic and damaged cells, efferocytosis also plays an important role in the resolution of inflammation by protecting tissues from harmful exposure to the inflammatory and immunogenic contents of dying cells (Kimani et al., 2014). It has been reported that defective clearance of apoptotic cells by macrophages occurs in both animals and humans with SLE (Tsokos et al., 2016;

Experimental mice

Female BALB/c mice (6–8 weeks old) were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China) and were housed in a pathogen-free conditions in a 12-h light and dark cycle. All procedures involving mice were approved by the institutional guidelines for animal care and were used according to the guidelines of the Animal Care Committee at Nanjing University. The skin on the right ears of the mice was treated topically, 3 times a week, with 1.25 mg of 5% imiquimod

Declaration of Competing Interest

All authors state that they have no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No.31872732, 31570909) and Key Research and Development Program of Jiangsu Province (No. BE2019706).

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